The Effect of Pimpinella Anisum and Origanum Vulgare Extracts Against Streptococcus Sanguinis, Streptococcus Mutans, and Streptococcus Salivarius

Statement of the Problem: There are global efforts for introducing a new herbal antimicrobial agent with minimal side effects. There are some reports about the antimicrobial properties of Pimpinella anisum and Oregano Vulgare. Purpose: In this study, the antimicrobial properties of Pimpinella anisum and Oregano Vulgare have been assessed. Material and Method: In this experimental in vitro study, the dental plaque samples were collected from children aged 3 to 5 years old who were referred to a private dental office with diagnosis of dental caries. After determination of the bacterial colonies of Streptococcus sanguinis, Streptococcus mutans and Streptococcus salivarius, the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of ethanolic and methanolic extracts of Pimpinella anisum and Oregano vulgare were measured by macrodilution and microdilution methods. Results: The mean MIC and MBC of Pimpinella anisum extract and Oregano vulgare extract and their combination against Streptococcus mutans, Streptococcus sanguinis, and Streptococcus salivarius were statistically different (p< 0.001). The combination of these extracts showed the lowest MIC and MBC. Conclusion: Hydroalcoholic extracts of the Pimpinella anisum and Oregano Vulgare were effective antibacterial agent against Streptococcus mutans, Streptococcus salivarius, and Streptococcus sanguinis so the combination of these two extracts showed the highest antibacterial properties on all the bacteria evaluated.


Introduction
The mouth environment can support the dental plaque formation [1]. Temperature, pH, saliva, and oxidationreduction (redox) reactions are the main factors related to plaque development [2][3][4]. Dental plaque is a bacterial biofilm, which is formed on different surfaces in the mouth. Dental plaque is the main causative factor for dental caries and periodontal diseases. Biofilm generation first starts with pellicle formation. The loose attachment of some bacteria creates micro colonies and ultimately, biofilm maturation can cause dental patholo-gies [5][6]. The development of a biofilm allows aggregation of cell colonies, which are increasingly resistant to antibiotics [7][8]. There are many different bacteria responsible for biofilms formation, including grampositive and gram-negative species [4,[8][9]. Origanum vulgare (O.vulgare) is a popular species of Origanum from the mint family (Lamiaceae) [10]. It is native to western, South-western Eurasia, and the Mediterranean region. In Austrian folk medicine, Oregano was used as a tea or as an ointment for gastrointestinal, respiratory tract, and nervous system diseases [10]. Over 60 differ-ent compounds have been extracted. Carvacrol and thymol are dominant compounds (80%) [11]. The antibacterial activity of carvacrol against several bacteria strains such as Escherichia coli and Bacillus cereus has been reported [12]. Natural biocidal agents such as thymol can decrease the possibility of bacterial resistance to common antibiotics such as penicillin [13].
At present, due to the indiscriminate use of antibiotics, different bacteria have developed drug resistance, so new researches are conducted to introduce novel antibacterial agents. Herbal agents or their ingredients are broadly evaluated [7]. Considering the properties and ingredients of P.anisum and O.vulgare, we decided to investigate their antibacterial effects and if possible introduce a new substance with antimicrobial activity with few side effects.
Sarac and Ugur [17] showed that the essential oils of Origanum onites L., O.vulgare were effective against some multiple antibiotic-resistant bacteria. Kermanshah et al. [18] showed that P.anisum had a growth inhibitory effect against Streptococcus mutans (S.mutans) and Lactobacillus rhamnosus. According to this study and other similar researches, and the antibacterial effect of these two herbal extracts, we decided to investigate the synergistic antibacterial effect of these extracts against dental plaque bacteria.

Materials and Method
In this experimental in vitro study, the dental plaque samples were collected from children aged 3-5 years old who were referred to a private dental office with diagnosis of dental caries.

Pimpinella Plant Extraction
Hydroalcoholic extraction of P.anisum and O.vulgare was obtained by "maceration" method. After preparing air-dried P.anisum, 50gr of its powder was weighted by a digital balance (Sartorius, Germany) and mashed.
1500cc of the solvent (half ethanol and half water) was added to them and shaken (IKA, Germany) for 48h and 90 cycles per minute until the composition reached homogeneity. The solution was filtered (Sartorius, Germany) and the solvent was vaporized by using a rotary evaporator (KNF, USA). The sterile extract was kept in the refrigerator for microbial evaluations.

Oregano Plants Extract
The plant was mixed with the solvent solution (20% methanol: 80% distilled water) by a ratio of 1:3(1mg powder+ 3ml solvent), and the mixture was uniformed by an electric blender for 30 minutes at room temperature. The solution was filtered for providing a transudation solution. It was dried using an incubator (Binder, Germany) at 50°C for 24 hours and the product was kept in a dry place until used. The ethanolic and methanolic extracts of P.anisum and O.vulgare were prepared.

Dental plaque sampling
The samples were collected from 3-5 years old children with dental caries. Dental caries was determined by cavitation on the tooth surface and evaluation of bitewing radiographs [20]. The white spot lesions and developmental grooves were excluded. A written consent form was obtained from the parents of the participants. The Ethics committee of Shiraz University of Medical Sciences has approved this study (IR.SUMS. REC. 1396.S160); also this evaluation has been conducted according to the Declaration of Helsinki (1975).
The participants did not have any systemic disease, especially one that could affect the rate of dental caries.
Samples were taken with a sterile toothpick from dental caries. The toothpick samples were kept in 1.0-mL reduced transport fluid vials for other processes. The plaque samples dilutions were plated onto MM10sucrose agar [21]. After 3 days of anaerobic incubation (85% N 2 , 10% CO 2 , and 5% H 2 ), the colonies were presumed to be Streptococcus sanguinis (S.sanguinis) and s.mutans, and Streptococcus salivarius (S. salivarius) was selected according to their colony morphology from MM10-sucrose agar [22][23].

Polymerase Chain Reaction (PCR)
The primer pairs of S. mutans and S. Sanguinis, and S.salivarius were used to detect them by PCR. These primers were 5-GqaGCACCACAACATTGGGAAGCTCA-GTT and 5-GGAATGGCCGCTAAGTCAACA-GGAT for

Statistical analysis
The data has been assessed in SPSS version 18. The p< 0.05 has been considered a significant point. Repeated measurement, ANOVA and sidak post hoc test was used in this study.

Results
Dental plaque of 60 participants (37 women and 23 men) with mean age of 4.65±1.12 years old, were collected  Figures 2 and 3).      Chaudhry et al. [24] reported antibacterial effect of aqueous extract of aniseed against S.salivarius, S.sanguinis, and S.mutans by disc diffusion method.

S.mutans (ATCC 25175) S.sanguinis (ATCC10556) S.salivarius (ATCC 9759) MIC (µg/ml) MBC (µg/ml) MIC (µg/ml) MBC (µg/ml) MIC (µg/ml) MBC (µg/ml)
The average diameter of the zone of inhibition of aniseed for S.salivarius was 14mm, which was the highest among these three species. In spite of more inhibitory effect of P.anisum extract on S.mutans in the present study, the report of more potent antibacterial effect of this extract against S.salivarius rather than S.sanguinis and S.mutans is considerable [24].
Sarac et al. [17]  Even a study introduced these two extracts as a more potent antimicrobial agent than beneficial probiotic bacteria (Lactobacillus) [12].
A research exhibited this potency in comparison to traditionally used essential oil in the dentistry clove oil [31]. phenol and thymol which are the most potent antibacterial agents [34][35].
In addition to some other properties of thymol and carvacrol, such as immunity enhancement against virus and tumor and anticancer activities, some mechanisms for their antibacterial activity have been proposed. These essential oils permeabilize and depolarize the cytoplasmic membrane, which causes a decrease in pH and consequently disturbs the proton motive force, reduces the intracellular ATP level and finally causes cell death [28,30].
Selecting proper solvent, evaluating the synergistic activity of using two effective extracts and using macro-

Conclusion
The hydroalcholic extract of P.anisum and O.vulgare on patient isolated dental caries bacteria (S.mutans, S.sanguinis, and S.salivarius) showed proper antimicrobial properties. The combination of these extracts exhibited very significant potency in antibacterial activity.